Methods and systems for reducing inflammation by neuromodulation of t-cell activity

ABSTRACT

Described herein are devices, systems and method of treating inflammation, including methods of treating a T-cell mediated disease. In particular, described herein are methods of treating inflammation including the steps of stimulating a subject&#39;s inflammatory reflex to inhibit the immune response and administering a T-cell modifying agent to modify the activity of splenic T-cells. Also described herein are systems for treating inflammation including an inflammatory reflex stimulation module and a T-cell response modifying module. The T-cell response modifying module typically modifies the response of splenic T-cells to enhance or otherwise regulate the effect of stimulation of the inflammatory reflex.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. Provisional patentapplication Ser. No. 61/072,603, titled “DEVICES AND METHODS FORNEUROMODULATION OF T-CELL ACTIVITY” filed on Mar. 31, 2008.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant(s) R01GM57226 and R01 GM62508s awarded by the National Institute for GeneralMedical Sciences of the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Cytokine production is counter-regulated by evolutionarily ancientmechanisms. The cholinergic anti-inflammatory pathway is ananti-inflammatory neural mechanism for suppressing cytokine release bythe immune system. It functions by signals carried via the vagus nervethat suppress cytokine release through a molecular mechanism thatrequires the alpha7 nicotinic acetylcholine receptor subunit (alpha7nAChR). Direct electrical (or mechanical) stimulation of the vagus nerveattenuates cytokine release and prevents tissue injury in experimentalanimals with cytokine mediated diseases, including endotoxemia,hemorrhagic shock, ischemia-reperfusion, sepsis, colitis, and arthritis.The functional integrity of this pathway is critical for modulating theinnate immune response to endotoxins, because eliminating the function,by either cutting the vagus nerve, or removing the alpha7 nAChR gene,renders animals exquisitely sensitive to otherwise innocuous quantitiesof endotoxins.

Early observations into the anatomic and functional basis of thecholinergic anti-inflammatory pathway implicate neural input to spleenas essential for vagus nerve mediated decreases of TNF duringendotoxemia. Electrical stimulation of the vagus nerve in the neckattenuates TNF mRNA and protein levels in spleen, a major source of TNFin endotoxemia. Surgical ablation of the vagus nerve branches to theceliac ganglion disrupts the TNF-suppressive activity of cervical vagusnerve stimulation. Innervation to the spleen is provided by the splenicnerve, a catecholaminergic nerve that originates in the celiac ganglion.Since the cholinergic antiiflammatory pathway requires alpha7 nAChRsignals, we examined how signals originating in vagus nerve reach theTNF-producing cells in spleen. Here we show that the splenic nerve isrequired for vagus nerve stimulation control of TNF production. Splenicnerve endings culminate adjacent to TNF producing macrophages andadjacent to T cells. Surprisingly, T cells are required for thefunctional integrity of the neural signals that inhibit TNF in spleen.As a result, we suggest methods and devices for treatment of disordersby stimulation of the inflammatory reflex (including the vagus nerve) incombination with modulation of T-Cells. Modulation of T-Cells, which may(in part) mediate the inflammatory reflex may help in furthercontrolling the inflammatory reflex.

This application is related to U.S. Pat. No. 6,610,713; U.S. patentapplication Ser. No. 10/990,938, titled “Inhibition of InflammatoryCytokine Production by Cholinergic Agonists and Vagus NerveStimulation,” filed Nov. 17, 2004; and U.S. patent application Ser. No.11/318,075, titled “Treating Inflammatory Disorders by Electrical VagusNerve Stimulation,” filed Dec. 23, 2005. Each of these patents andpending applications is herein incorporated by reference in itsentirety.

SUMMARY OF THE INVENTION

Described herein are devices, systems and method of treating aninflammatory response, including a method of treating a T-cell mediateddisease. For example, described herein is a method of treating aninflammatory response comprising: stimulating a subject's inflammatoryreflex to inhibit the immune response; and administering a T-cellmodifying agent.

Inflammatory responses to pathogens and tissue injury are preciselycontrolled to prevent excessive tissue injury. Signals in thecholinergic anti-inflammatory pathway, in which efferent vagus nervesignals inhibit cytokine production, require the alpha7 nicotinicacetylcholine receptor subunit (alpha7 nAChR), which binds acetylcholineand suppresses the nuclear translocation of NFkB. Despite the importanceof this pathway in controlling cytokine production, the cellular targetof the neural signals during endotoxemia was previously unknown. Here,immunohistochemical staining of spleen during endotoxemia revealed thatsplenic nerve endings terminate adjacent to discrete macrophagepopulations producing TNF, and next to T cells that produceacetylcholine. Application of either vagus nerve stimulation, oradministration of nicotine, an alpha7 agonist, significantly attenuatesTNF production by the subpopulation TNF-producing splenic macrophages.Surprisingly, however, vagus nerve stimulation of nude mice failed toinhibit TNF production. Administration of nicotine to nude mice didsignificantly inhibit TNF production, indicating that T cells areessential for the neural, but not molecular activation of thecholinergic anti-inflammatory pathway. Together these results indicatethat T cells are required for regulation of TNF by vagus nerve signalsto spleen, and identify a previously unknown role for T cells in thefunction of the cholinergic anti-inflammatory pathway.

In the methods, systems and devices described herein, the stimulation ofthe subject's inflammatory reflex may be electrical stimulation,mechanical stimulation, or any other appropriate method of stimulation.In particular, long-term stimulation methods (e.g., non-contact,non-desensitizing methods) may be preferred.

Any appropriate T-cell modifying agent may be used with the methods,systems and devices described herein. For example, the T-Cell modifyingagent may be selected from the group consisting of: glucocorticoids,antibody agents, peptide agents, drugs, and pro-drugs.

The step of stimulating the subject's inflammatory reflex may comprisestimulating the subject's vagus nerve (and/or afferent or efferents ofthe vagus nerve). In some variations the step of stimulating thesubject's inflammatory reflex comprises stimulating one or more of: thevagus nerve, the splenic nerve, the hepatic nerve and the trigeminalnerve, and their afferents and efferents.

The step of administering a T-cell modifying agent may comprisessystemic administration of the T-cell modifying agent, or localadministration of the T-cell modifying agent (e.g., to the spleen orgeneral splenic region). A T-cell modifying anent may be administered atany time in the method, for example, before, during and/or afterstimulating the subject's inflammatory reflex.

In some variations, the step of stimulating the inflammatory reflexcomprises repeatedly and periodically stimulating the subject'sinflammatory reflex. For example, a subject's vagus nerve may bestimulated every 4 hours, every 8 hours, every 12 hours, every 24 hours,every 2 days, etc. The method may also included additionaladministration of T-cell modifying agents. For example, a T-cellmodifying agent may be applied at various times during the treatmentmethod.

Also described herein are methods of treating T cell-mediated diseasescomprising the steps of identifying a patient suffering from a conditionmediated by T-cell cells, stimulating the subject's inflammatory reflex,and administering a T-cell modifying agent. The T-cell mediated diseasemay be selected from the group consisting of: transplant rejection,rheumatoid arthritis, Psoriasis, or multiple sclerosis.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference in their entirety to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the loss of inhibition the inflammatory reflex (measured byserum TNF) after cutting the splenic nerve (right side) compared tocontrol (sham, left side). Inhibition of the inflammatory reflex iscaused by VNS (vagus nerve stimulation), as previously described.

FIGS. 2A and 2B shows immunohistochemical staining for synaptophysin(SYN) either before (FIG. 2A) or after (FIG. 2B) cutting the splenicnerve.

FIGS. 2C and 2D shows immunohistochemical staining for catecholaminergicneurons (CAT) before (FIG. 2C) or after (FIG. 2D) cutting the splenicnerve.

FIGS. 3A-3D show immunohistochemical staining with specific markers ofmacrophage subpopulations and TNF. MZM (marginal zone macrophages) andMMM (marginal metallophilic macrophages) and red pulp macrophages areindicated from this splenic section. TNF staining overlapped withmarginal zone and the red pulp regions.

FIGS. 4A-4D are immunohistochemical staining of rats (through thespleen) after cutting the splenic nerve (FIGS. 4C and 4D) and insham-operated rats, whose splenic nerve was left intact (FIGS. 4A and4B); all rats were first subjected to vagus nerve stimulation andendotoxemia. Spleen sections were stained for TNF expression.

FIGS. 5A-5B show spleen sections stained for synaptophysin (SYN), TNF,and red pulp macrophages (RPM) in sham-operated (control) rats.

FIGS. 5C and 5D show spleen sections stained for synaptophysin (SYN) andCD3 (a T cell marker).

FIGS. 5E and 5F show spleen sections expressing eGFP placed under thecontrol of endogenous ChAT transcriptional regulatory elements(eGFT-ChAT) and stained for synaptophysin (SYN).

FIG. 6A shows the effect of serum TNF levels in wild-type (left) andnude (right) mice (mice devoid of T cells) after vagus nerve stimulationduring endotoxemia. “SHAM” mice were not stimulated.

FIG. 6B shows the effect of serum TNF levels in wild-type (left) andnude (right) mice during endotoxemia after application of either saline(PBS) or nicotine.

FIG. 7A shows the effect of a cholinesterase inhibitor, Paraoxon, on TNFproduction by LPS-stimulated spleen cells, indicating a dose-dependentinhibition of TNF. FIG. 7B indicates that here is no significanttoxicity correlated to these levels of Paraoxon.

FIG. 8A illustrates a schematic of a general system for modulating aninflammatory reflex. FIG. 8B is a schematic illustration of onevariation of system for modulating an inflammatory reflex.

DETAILED DESCRIPTION OF THE INVENTION

To study whether TNF-inhibiting signals in the vagus traverses thesplenic nerve, the splenic nerve was surgically ablated in rats exposedto endotoxemia and vagus nerve stimulation. Splenic neurectomy preventedthe TNF-suppressing activity of vagus nerve stimulation, indicating thatthe neural signals originating in the cervical vagus nerve traverse thesplenic nerve (FIG. 1). Complete ablation of the splenic nerve wasconfirmed, because immunohistochemical staining for synaptophysin, asynaptic vesicle protein and marker of nerve terminals, was absent afterthe splenic nerve was cut (FIGS. 2A and 2B). Moreover, and as expected,review of sections stained using glyoxylic acid to revealcatecholaminergic neurons revealed that it was completely depleted inanimals subjected to splenic neurectomy (FIGS. 2C and 2D). In agreementwith previous studies, we observed synaptophysin-positive staining inthe regions of the central artery, white pulp, red pulp, and spleniccapsule. Sequential sections showed synaptophysin staining following asimilar distribution pattern to that of the neural network visualizedwith glyoxylic acid.

To continue studying the functional and cellular anatomy of thesenerves, it was necessary to characterize the cell source of spleen TNFduring endotoxemia. Previous evidence suggested that the major source ofTNF in endotoxemia is cells of the rethiculoendothelial system (Cano,G., A. F. Sved, L. Rinaman, B. S. Rabin, and J. P. Card. 2001.Characterization of the central nervous system innervation of the ratspleen using viral transneuronal tracing. J. Comp Neurol. 439:1-18.1),but the specific cell type was unknown. Accordingly, sections studied byimmunohistochemistry revealed that TNF production occurred in themarginal zone and the red pulp regions, the areas where spleenmacrophages reside. Immunohistochemistry with specific markers ofmacrophage subpopulations revealed the TNF-producing cells to be themarginal metallophilic macrophages, the marginal zone macrophages, andthe red pulp macrophages (FIGS. 3A-3D). We did not observe TNF-positivestaining in either T lymphocytes or dendritic cells. These findingsindicated that macrophages in the marginal zone and red pulp are themajor source of spleen TNF in endotoxemia. In order to confirm thefunctional connection between vagus nerve signals and theseTNF-producing macrophages, rats were subjected to vagus nervestimulation and endotoxemia. TNF expression in spleen was significantlyabolished by vagus nerve stimulation (FIGS. 4A-4D). Together theseresults indicate that signals originating in the vagus nerve attenuateTNF in spleen via the splenic nerve.

We next undertook to understand the cellular basis for this control ofcytokine production by specific nerves in discrete populations ofmacrophages. We performed a systematic search for points of contactbetween the splenic nerve endings and the TNF producing macrophages.Triple staining of spleen sections from sham-operated rats revealednerve terminals adjacent to, and apparently contacting, theTNF-producing macrophages (FIGS. 5A and 5B). Nonetheless, these pointsof contact were difficult to visualize, and largely limited to the redpulp macrophages. Most important, not all of the TNF producing cellsreceived neural input. Instead, it was clear that the major cellscontacting the neurons were the T cell in the white pulp. Serialsections through white pulp areas revealed that synaptophysin-positiveneurons provided abundant contact to T cells (FIGS. 5C and 5D). Previousinvestigators have shown that catecholaminergic neurons contact T cellsin spleen, and that T cells express choline acetyltransferase, theenzyme required for acetylcholine production. BAC-transgenic mice, inwhich eGFP was placed under the control of endogenous ChATtranscriptional regulatory elements were used. Examination of eGFP-ChATin spleen confirmed that T cells are the major source of acetylcholinesynthesis in spleen. Moreover, nerve terminals made contact with some ofthese eGFP-ChAT-positive cells (FIGS. 5E and 5F).

Accordingly, we reasoned that the T cells might participate in theneural regulation of TNF in the spleen. Consistent with this, wediscovered that vagus nerve stimulation of nude mice (mice devoid of Tcells) failed to inhibit TNF production during endotoxemia (FIG. 6A). We(and others) have previously shown that the alpha7 nAChR is essentialfor the functional integrity of the cholinergic anti-inflammatorypathway. Administration of nicotine to nude mice significantly inhibitedthe production of TNF (FIG. 6B) indicating the absence of T cells doesnot impair the ability of the TNF producing macrophages to respond tothe alpha7 nAChR agonist. Thus, we have shown the surprising result thatT cells provide a critical role in transducing neural signals from thesplenic nerve to the TNF-producing macrophages to control cytokineproduction via the cholinergic anti-inflammatory pathway.

These surprising results described herein expand our knowledge of theanatomical basis of the cholinergic anti-inflammatory pathway by showingthat macrophage subpopulations in spleen produce TNF in endotoxemia, andthat electrical stimulation of the vagus nerve controls TNF productionby these cells via a mechanism that is dependent on an intact splenicnerve.

Previously, we found that vagus nerve stimulation attenuates spleen TNF,an effect that is dependent on the celiac branches of the vagus nerve.However, neuronal tracing studies and the lack of splenic cholinergicfibers indicate that primary vagus nerve fibers do not innervate thespleen. Our results show that suppression of TNF by vagus nervestimulation requires an intact splenic nerve, a finding that indicates afunctional connection between the vagus nerve and splenic TNF-producingcells. The spleen is innervated by nerve fibers that originate in theceliac ganglion. Efferent fibers of the vagus nerve, through its celiacbranches, terminate in synaptic-like structures around principal cellsof the celiac ganglion. Our data raise the possibility that ananti-inflammatory signal conveyed by the vagus nerve attains splenicimmune cells through a system of two serially-connected neurons: onepreganglionic that originates in the dorsal motor nucleus of the vagusembodied in the vagus nerve, and other postganglionic that originates inthe celiac ganglion whose axons travel along the splenic nerve.

The splenic nerve, mainly composed of catecholaminergic fibers, issupplied by preganglionic sympathetic neurons located in theintermediolateral column of the thoracic spinal cord and theparavertebral ganglia (1). Our results indicate that the vagus nervecommunicates with the splenic nerve. Vagal regulation of postganglioniccatecholaminergic fibers has been demonstrated previously. In dogs,electrical stimulation of the cervical vagus nerve decreases pancreaticnorepinephrine release induced by electrical stimulation of thoracic(catecholaminergic) nerves (2). In rats, cervical vagotomy increasessplenic nerve discharge induced by IL-1β, suggesting that the vagusnerve exerts a tonic inhibitory control over splenic nerve activity (3).To our knowledge, preganglionic projections to postganglionic neurons inthe celiac ganglion have not been characterized. For instance, it isunclear whether the splanchnic nerve (preganglionic sympathetic fiber)and the vagus innervate the same postganglionic neurons in the celiacganglion.

Vagus nerve stimulation attenuates TNF mRNA and protein levels inspleens of endotoxemic animals but the target cell of this effect wasunknown. Here we found that vagus nerve stimulation attenuated TNF inmacrophages of the marginal zone and the red pulp. An earlier studyusing the perfused rat spleen ex vivo showed that electrical stimulationof the splenic nerve induces norepinephrine release from spleen, andattenuates LPS-induced TNF through a beta-adrenergic dependent mechanism(4). Given that electrical stimulation of the vagus nerve requires anintact splenic nerve in order to suppress TNF, it is plausible thatvagus nerve stimulation induces release of norepinephrine fromcatecholaminergic splenic nerve terminals, whose presence has beenpreviously revealed in the marginal zone and red pulp. Futureexperiments will aim to determine the effect of vagus nerve stimulationon the release of catecholamines and other neurotransmitters orneuropeptides (e.g. neuropeptide Y) in spleen.

One issue in the cholinergic anti-inflammatory pathway not addressed inthis study is the anatomic location of the nicotinic acetylcholinereceptor alpha7 subunit, which is required for vagus nerve control ofTNF production. In vivo, we have observed that alpha7 knockout mice areinsensitive to the TNF-suppressive effect of vagus nerve (5). In vitro,acetylcholine and other cholinergic agonists attenuate LPS-induced TNFin human and mouse macrophages, as well as in mouse splenocytes, throughan alpha7-dependent mechanism (5,6). In view of the present findings itis possible that the alpha7 requirement observed in vivo is related toits functioning in autonomic ganglia, the alpha7 nicotinic subunit ofthe acetylcholine receptor is expressed in autonomic ganglia where itmediates fast synaptic transmission, and its expression has beendocumented in the superior cervical ganglion and the celiac ganglion(7,8). It is possible that acetylcholine released by the vagus nerveacts upon alpha7 expressed in neurons of the celiac ganglion to modulatesplenic nerve function. Nevertheless, alpha7 involvement in cholinergicsignaling in spleen, which contains and produces acetylcholine (9,10)and alpha7-expressing immune cells (11), has not been ruled out.

Here, we have found that several macrophage subpopulations areresponsible for splenic TNF production in endotoxemia, and that theircytokine-producing capability is amenable to modulation by electricalstimulation of the vagus nerve, through a mechanism dependent on anintact splenic nerve. The spleen is a lymphoid organ where severalleukocyte subtypes converge to initiate innate and adaptive immuneresponses, and splenic nerve endings are located in close proximity tomacrophages and lymphocytes. Our finding that vagus nerve stimulationmodulates splenic TNF production through the splenic nerve, suggest thatvagus nerve stimulation could modulate adaptive immunity as well.Altogether, these findings extend our knowledge of the anatomical basisthat allows control of cytokine production in the spleen by the vagusnerve.

MATERIALS AND METHODS Animals

Adult male BALB/c mice 8 to 12 weeks-old (20-25 g; Taconic) and adultmale Sprague Dawley rats 8 to 12 weeks-old (250-300 g, Charles RiverLaboratories) were housed at 25° C. on a 12-hour light/dark cycle, andlet acclimatize for one week before experiments were conducted. Waterand regular rodent chow were available ad libitum. Experiments wereperformed under protocols approved by the Institutional Animal Care andUse Committee of the Feinstein Institute for Medical Research, NorthShore-LIJ Health System.

Antibodies

Antibodies and dilutions used for immunofluorescence were as follows:Marginal zone macrophages: Biotinylated rat anti-mouse SIGN-R1 (cloneER-TR9, BMA Biomedicals); marginal metallophilic macrophages:Biotinylated rat anti-mouse sialoadhesin (clone MOMA-1, BMABiomedicals); red pulp macrophages: Biotinylated rat anti-mouse F4/80(clone A3-1, Serotec); neutrophils: Rat anti-mouse Gr-1 (clone RB6-8C5,R&D Systems); rat marginal zone macrophages: Mouse anti-rat CD169 (CloneED3, Serotec); rat red pulp macrophages (Clone ED2, Serotec). Goatanti-mouse TNF (R&D Systems), and goat anti-rat TNF (R&D Systems).Secondary reagents: Cy3-conjugated Affinipure donkey anti-goat IgG(Jackson immunoresearch Laboratories); FITC-conjugated Affinipure donkeyanti-rabbit IgG (Jackson Immunoresearch Laboratories); Alexa Fluor568-conjugated streptavidin (Molecular Probes); FITC-conjugated avidin(Molecular Probes).

Endotoxemia

Endotoxin (LPS from E. coli, 0111:B4, Sigma-Aldrich) was injected toanimals (10 mg/kg, i.p. corresponding to a LD75 dose). Blood and spleenswere harvested 10, 30, 60 or 120 minutes after LPS administration. Weused ELISA to determine TNF concentration in serum (R&D Systems).Spleens were either snap frozen for further immunofluorescence analysisor disrupted in PBS plus protease inhibitor cocktail (Complete mini,Roche) with a tissue homogenizer (Polytron 3100, Kinematica). TNFcontent in spleen tissue was determined by ELISA and normalized toprotein concentration (Protein Assay, BioRad). In some experiments, PBSor nicotine (Sigma-Aldrich) was injected 30 minutes (2 mg/kg, i.p.)prior to endotoxin administration. Animals were then euthanized 60minutes later and spleens were harvested for TNF immunofluorescence orTNF quantification in tissue homogenates.

Electrical Stimulation of the Vagus Nerve

Male Sprague Dawley rats were anesthetized with urethane (4 g, i.p.) andxylazine (15 mg/kg, i.m.). Vagus nerve stimulation was performed asdescribed previously (11). Briefly, a bipolar platinum electrode(Plastics One) was placed across the isolated cervical vagus nerve.Electrical stimulation (1V, 2 ms, 5 Hz) was generated by a stimulationmodule (STM100A) under the control of the Acknowledge software (BiopacSystems). Rats underwent 10 minutes of vagus nerve stimulation beforeand after endotoxin injection. In rats subjected to sham surgery, thevagus nerve was only exposed.

Immunofluorescence

All samples were fresh-frozen with dry ice, embedded in O.C.T. compound(Tissue-Tek) and kept at −20° C. until processing. Ten μm-thick spleenslices were cut using a cryostat and mounted on glass slides andair-dried for five minutes. The tissue was then permeabilized withPBS-saponin 0.1% for 30 minutes. All incubation periods were performedat room temperature in a humid chamber. The primary antibodies werediluted in PBS-saponin 0.1% at the following concentrations: TNF (1:6dilution), MOMA-1 (1:1000 dilution), ER-TR9 (1:100 dilution), F4/80(1:50). After a 2-hour incubation period, the slides were washed 3 timesin washing buffer (PBS, tween 20 0.02%), incubated with avidin-FITC andCy3-labeled rat anti-goat IgG, both diluted 1:250 in PBS-saponin 0.1%for 30 minutes. Finally, the slides were washed, dried, mounted(Vectashield) and observed through a Zeiss Axiovert 20 invertedmicroscope. Images were analyzed using the AxioVision V5 software (CarlZeiss).

METHODS OF TREATMENT

In general, an inflammatory response may be treated based on this newdiscovery. In particular, an inflammatory reflex may be treated bystimulating a subject's inflammatory reflex to inhibit the immuneresponse, and administering a T-cell modifying agent.

The inflammatory reflex may include the vagus nerve, the splenic nerve,the hepatic nerve and the trigeminal nerve, and their afferents andefferents. For example, Tracey et. al., have previously reported thatthe nervous system regulates systemic inflammation through a vagus nervepathway. This pathway may involve the regulation of inflammatorycytokines and/or activation of granulocytes. Thus, it is believed thatappropriate modulation of the vagus nerve may help regulateinflammation. In some variations the inflammatory reflex may be limitedto one or more of these nerves (or its afferents or efferents), such asthe vagus nerve.

The inflammatory reflex may be stimulated by any appropriate method,particularly electrical or mechanical stimulation. Other forms ofstimulation include magnetic stimulation, thermal stimulation, etc. Whenelectric and mechanical stimulation is used, this stimulation may beperformed without desensitizing the inflammatory reflex. For example,the stimulation may be performed by an electrode or actuator that doesnot directly contact the nerve. Any appropriate stimulation may be used,particularly stimulation which results in a long-lasting (andrepeatable) inhibition of inflammation, including cytokine levels.Examples of such stimulation are provided in the documents incorporatedby reference, but may include stimulation at extremely low duty-cyclesuch as stimulation for less than 5 minutes once every 6 hours, every 12hours, every 24 hours, or longer. An exemplary electrical stimulationmay be stimulation at in the range of 10 mV to 5 V at a frequency of 0.1Hz to 100 Hz, with a duration of stimulation between from 1 ms to 10min.

In some variations, the stimulation is modified by the addition of theT-cell modification agent. For example, stimulation may be reduced inintensity (e.g., voltage, pressure, etc.) or in duration (e.g.,frequency within stimulation pulse) or in regularity (e.g., durationbetween stimulation pulses), or the like. Feedback (open loop or closedloop) may be used to set the intensity, duration, and/or regularity ofstimulation.

The stimulation of the inflammatory pathway may be provided by anexternal device, an internal (e.g., implanted) device, or a devicehaving both internal and external components. For example, a stimulationdevice may include a non-contact electrode that does not contact a nerveof the inflammatory reflex, an energy source to apply energy to theelectrode(s) and a controller. The controller may control theapplication of energy to the electrode(s). For example, the controllermay initially cause the stimulator to apply stimulation once or twice aday for 1 minute of 0.1 V stimulation having a frequency of 1 Hz and aduration of 50 ms. The stimulator may be part of a system for treatingthe inflammatory reflex, which may also include one or more sensors,including feedback sensors. Feedback sensors may help control thesystem, including the application of stimulation to the inflammatorypathway, and/or the administration of one or more T-cell modulatingagents.

Agents that modulate T-cells may include immunosuppressants andimmunomodulators. For example, agents such as glucocorticoids (e.g.,dexamethasone, hydrocortisone, prednisone, prednisolone, etc.), antibodyagents (e.g., antibodies or fragments directed against the CD3 moleculeof the T-cell antigen receptor complex, such as Muromonab CD3(0KT-3),etc.), peptide agents (e.g., immunosuppressive agents includingCyclosporine, etc.). Drugs (e.g., tacrolimus, Aspirin-like drugs (ALD),etc.) and “pro-drugs” (such as Azathioprine, Mycophenolate Mofetil,Methotrexate, etc.) are also included. Agents may act directly onT-cells or indirectly to affect T-cells. For example, agents mayinteract with receptors on the surface of the T-cell (e.g., caspase 8inhibitor c-FLIP(L), and related agents), and may modulate (e.g.,inhibit or enhance) T-cell response and/or proliferation. The examplesprovided above are not exhaustive, and any agent which may be used tomodulate T-cells (e.g., T-cell activity or proliferation) may be used.

The modulation of T-cells may include potentiating activity, includingpotentiating acetylcholine (ACh) activity. For example, a T-cellmodifying agent may include an agent that potentiates release ofacetylcholine. Such agents may include small molecules, antibodies,peptides, or the like. For example, anti-CD11a antibodies have beenshown in some circumstances to enhance or trigger release ofacetylcholine (e.g., T. Fujii, K. Masuyama, K. Kawashima, “Simvastatinregulates non-neuronal cholinergic activity in T lymphocytes viaCD11a-mediated pathways” J Neuroimmunol. October 2006; 179(1-2):101-7.Epub Jul. 10, 2006). In some variations, the T-cell modifying agent maypotentiate the stability of ACh, for example, by preventing uptakeand/or breakdown of ACh. For example, the activity of the T-cell may bemodulated by inhibiting acetylcholine esterase. Alternatively, theactivity of the T-cell may be modulated by destabilizing (e.g.,removing) acetylcholine; for example, by adding esterase.

In general, the methods and systems described herein are directed to thetreatment of inflammation by modulating T-Cell activity, in somevariations the activity of acetylcholine is directly modulated, so thatsources of acetylcholine or agonists and antagonists implicated in theacetylcholine signaling pathway (e.g., acetylcholine receptors, etc.)including non-T-cell sources may also be manipulated as part of themethods and systems described herein. For example, the method mayinclude a method of coordinating stimulation of the subject'sinflammatory reflex to inhibit the inflammatory response andadministering an agent that modulates or potentiates splenicacetylcholine, which may include acetylcholine released by T-cells.

Agents or drugs may be delivered systemically to the subject, or theymay be delivered locally. For example, agents may be delivered orally,intraveneously, intramuscularly, etc. In some variations, the agent maybe delivered by an implant. For example, the implant may be part of asystem for stimulating (e.g., electrically, mechanically, etc.) theinflammatory reflex. In some variations the T-cell modifying agent isapplied locally to the spleen, including the region communicating withthe spleen (e.g., splenic region).

The method of treating an inflammatory response may be used to treat anyinflammatory response, including an inflammatory disorder. Anyinflammatory disorder may be treated in this manner. For example,disorders mediated by a cytokine response may be treated as describedherein.

Examples of inflammatory disorders may include (but are not limited to):transplant rejection, rheumatoid arthritis, Psoriasis, or multiplesclerosis. Inflammatory disease that include T-cell mediated diseasesmay include: inflammatory bowel disease, systemic lupus erythematosis,rheumatoid arthritis, juvenile chronic arthritis, spondyloarthropathies,systemic sclerosis (scleroderma), idiopathic inflammatory myopathies(dermatomyositis, polymyositis), Sjogren's syndrome, systemic vaculitis,sarcoidosis, autoimmune hemolytic anemia (immune pancytopenia,paroxysmal nocturnal hemoglobinuria), autoimmune thrombocytopenia(idiopathic thrombocytopenic purpura, immune-mediated thrombocytopenia),thyroiditis (Grave's disease, Hashimoto's thyroiditis, juvenilelymphocytic thyroiditis, atrophic thyroiditis), diabetes mellitus,immune-mediated renal disease (glomerulonephritis, tubulointerstitialnephritis), demyelinating diseases of the central and peripheral nervoussystems such as multiple sclerosis, idiopathic polyneuropathy,hepatobiliary diseases such as infectious hepatitis (hepatitis A, B, C,D, E and other nonhepatotropic viruses), autoimmune chronic activehepatitis, primary biliary cirrhosis, granulomatous hepatitis, andsclerosing cholangitis, inflammatory and fibrotic lung diseases (e.g.,cystic fibrosis), gluten-sensitive enteropathy, Whipple's disease,autoimmune or immune-mediated skin diseases including bullous skindiseases, erythema multiforme and contact dermatitis, psoriasis,allergic diseases of the lung such as eosinophilic pneumonia, idiopathicpulmonary fibrosis and hypersensitivity pneumonitis, transplantationassociated diseases including graft rejection and graft-versus hostdisease, Psoriasis and other skin disorders.

The step of administering a T-cell modifying agent may be performed atany point in the methods described herein, as appropriate. For example,in some variations, the T-cell modifying agent is administeredconcurrent with the stimulation of the inflammatory reflex. In somevariations the T-cell modifying agent is given before stimulation of theinflammatory reflex (e.g., seconds, minutes, hours, or days before). Insome variations, the T-cell modifying agent is given after stimulationof the inflammatory reflex (e.g., seconds, minutes, hours, or daysafter). Multiple administrations of T-cell modifying agents may begiven. In some variations, multiple T-cell modifying agents may be giveneither simultaneously or at different times.

The benefit of both administration of T-cell modifying agents andstimulation of the inflammatory reflex may result in an enhanced effectthat would otherwise be seen with either stimulation or administrationalone. This may allow a decreased amount of stimulation oradministration to be provided. In some instances the effect may befurther enhanced by the combination. For example, a T-cell modifyingagent that does not significantly inhibit inflammation may be effectiveto inhibit an inflammatory response when combined with stimulation ofthe vagus nerve or some other component of the inflammatory reflex.

In some variations, an agent that modifies T-cell activity may beapplied to inhibit the inflammatory reflex, which may otherwise bestimulated. For example, an agent that inhibits T-cell activity (and/orproliferation) may be provided to inhibit the inflammatory reflex, whichmay enhance inflammation, or may remove an inhibition on inflammation.

A method of treating a T cell-mediated diseases may include the steps ofidentifying a patient suffering from a condition mediated by T-cellcells, stimulating the subject's inflammatory reflex, and administeringa T-cell modifying agent.

A “T-cell mediated” disease means a disease in which T cells directly orindirectly mediate or otherwise contribute to the morbidity in a mammal.The T cell mediated disease by be associated with cell mediated effects,lymphokine mediated effects, etc. and even effects associated with Bcells if the B cells are stimulated, for example, by the lymphokinessecreted by T cells.

Compounds that modify the Acetylcholine response of the T-cells are oneclass of T-cell modifying agents that may be used herein. For example, aT-cell modifying agent may include a material that effects either therelease, the update, or the breakdown of acetylcholine in the spleen,and particularly by the T-cells in proximity to the splenic nerveendings. For example, FIGS. 7A and 7B illustrates the effect of Paraoxonon spleen cells. Paraoxon is a cholinesterase inhibitor. FIG. 7A showsthat increasing the amount of ACHE inhibitor in whole spleen cellcultures stimulated with LPS results in a blunted TNF response. Asmentioned above, the source of TNF in LPS-stimulated spleen cellcultures is thought to be the splenic macrophage population. Based onthe data provided herein (e.g., FIG. 7A), there is an endogenous sourceof acetylcholine in the whole spleen cell preparation, and if theactivity of ACh is increased in these cultures by inhibiting ACHE, theTNF response is effectively blunted.

Although the spleen is innervated by catecholaminergic nerve fibers,neural cholinergic input is absent. However, the spleen containsacetylcholine and releases it upon electrical stimulation of the splenicnerve. In order to characterize the source of acetylcholine in spleen,we used transgenic mice in which enhanced green fluorescent protein(eGFP) is expressed under control of transcriptional regulatory elementsof choline acetyltransferase, the enzyme that synthesizes acetylcholine.eGFP was detected in spleen B and T cells, some of which were located inclose proximity to nerve endings in the parenchyma of the white pulp.Paraoxon, a cholinesterase inhibitor that enhances cholinergictransmission by preventing hydrolysis of acetylcholine, dose-dependentlyattenuated TNF levels in spleen cell suspensions stimulated with LPSwithout affecting cell viability, suggesting that acetylcholine derivedfrom lymphocytes is involved in regulation of spleen TNF.

For example, FIG. 7A illustrates the effect of increasing concentrationsof Paraoxon on TNF production by LPS-stimulated spleen cells of BALB/c18 week old mice. As mentioned above, as the amount of Paraoxon isincreased (thereby increasing the amount of acetylcholine by increasingthe inhibition of AChE), the greater the inhibition of TNF. In thisexample, differing concentrations of Paraoxon were included in variousindividual wells (having 5×10⁶ cell/well) with a total volume of 250microliters/well, and stimulated for 4 hours with LPS (500 ng/mL). FIG.7B illustrates that for the concentrations of Paraoxon used in FIG. 7A,there was no significant loss of viability, so the effect was not likelydue to a reduction in macrophages or other cells producing TNF, but isinstead the result of inhibition of TNF production due to acetylcholine.

Thus, inflammation may be inhibited by stimulating the inflammatoryreflex and by administering a T-cell modifying agent, as mentionedabove. In some variations, the inflammatory response may be inhibited bystimulating a subject's inflammatory reflex and administering a T-cellmodifying agent that enhances the ACh component of the inflammatoryreflex (either before, during, or after stimulation of the inflammatoryreflex).

In some variations a system for modulating an inflammatory response bymodifying a subject's T-cells includes one or more components forstimulating a portion of the inflammatory reflex (e.g., the vagusnerve), and one or more components for applying a T-cell modifyingagent. The component for stimulating the T-cell modifying agent may bean implant that releases one or more compounds that modify the activityof a T-cell. For example, the system may include an implantable drugdelivery system that releases a compound. The implantable drug deliverysystem may be configured to be implanted in or near the spleen, so thatthe drug may be delivered to the spleen, and particularly near theterminals of the splenic nerve adjacent to T-cells and/or macrophages.In general the component for applying a T-cell modifying agent (e.g., adrug-delivery system or sub-system) and the component for stimulating aportion of the inflammatory reflex (e.g., a vagus nerve stimulator)communicate so that the stimulation of the inflammatory reflex may becoordinated with the modification of the T-cells. Thus, the system maybe configured so that the T-cell modifying agent is applied eitherbefore or during (or in some cases, after) the stimulation of one ormore portion of the inflammatory reflex.

FIG. 8A illustrates a schematic of one variation of a general system formodulating an inflammatory reflex, as described herein, including aninflammatory reflex stimulation module 801 and a T-cell modificationmodule 803. These two modules may communicate (e.g., wirelessly or viadirect connection 805). In some variations, the system includes acontroller for coordinating the activity of these modules. FIG. 8B is aschematic illustration of one variation of this system. In FIG. 8B, theinflammatory reflex stimulation module includes an implantable electrodefor stimulating the vagus nerve 811, a controller for coordinating thestimulation from the electrode 813, and a power supply 815 for providingthe power (e.g., battery). The power supply and/or controller may beimplantable with the electrode, or they may be external. The T-cellmodification module includes an implantable drug depot 821, and adelivery controller 823 for controlling the release of drug from theimplantable drug depot 823. The drug depot is configured to be implanted(e.g., in or near the spleen) and to deliver drugs to reach the T-cells(or a subpopulation of the T-cells) in the spleen and modify theiractivity. Any of the agents mentioned herein for modifying the T-cellresponse may be used. The drug depot may be powered or passive. Thedelivery controller for the drug depot may communicate directly orindirectly (e.g., wirelessly) 825 with the controller for the vagusnerve stimulator. Any of these systems may include a controller forcoordinating the stimulation between the inflammatory reflex stimulationmodule and the T-cell modifying agent module.

While the devices, systems, and methods of using them have beendescribed in some detail here by way of illustration and example, suchillustration and example is for purposes of clarity of understandingonly. It will be readily apparent to those of ordinary skill in the artin light of the teachings herein that certain changes and modificationsmay be made thereto without departing from the spirit and scope of theinvention.

REFERENCES

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1. A method of treating an inflammatory response, the method comprising:stimulating a subject's inflammatory reflex to inhibit the immuneresponse; and administering a T-cell modifying agent.
 2. The method ofclaim 1, wherein the step of stimulating the subject's inflammatoryreflex comprises electrically stimulating the subject's inflammatoryreflex to inhibit the immune response
 3. The method of claim 1, whereinthe step of stimulating the subject's inflammatory reflex comprisesmechanically stimulating the subject's inflammatory reflex to inhibitthe immune response.
 4. The method of claim 1, wherein the T-cellmodifying agent is selected from the group consisting of:glucocorticoids, antibody agents, peptide agents, drugs, and pro-drugs.5. The method of claim 4, wherein the T-cell modifying agent comprisesan agent that potentiates splenic acetylcholine.
 6. The method of claim5, wherein the T-cell modifying agent comprises an AChE inhibitor. 7.The method of claim 1, wherein the step of stimulating the subject'sinflammatory reflex comprises stimulating the subject's vagus nerve. 8.The method of claim 1, wherein the step of administering the T-cellmodifying agent comprises systemic administration of the T-cellmodifying agent.
 9. The method of claim 1, wherein the step ofadministering the T-cell modifying agent comprises local administrationof the T-cell modifying agent to the splenic region.
 10. The method ofclaim 1, wherein the step of administering the T-cell modifying agentcomprises administering the T-cell modifying agent before stimulatingthe inflammatory reflex.
 11. The method of claim 1, wherein the step ofstimulating the inflammatory reflex comprises repeatedly andperiodically stimulating the subject's inflammatory reflex.
 12. A methodof treating T cell-mediated diseases comprising: identifying a patientsuffering from a condition mediated by T-cell cells; stimulating thesubject's inflammatory reflex; and administering a T-cell modifyingagent.
 13. The method of claim 12, wherein the T-cell mediated diseaseis selected from the group consisting of: transplant rejection,rheumatoid arthritis, Psoriasis, or multiple sclerosis.
 14. A system fortreating the inflammatory reflex, the system comprising: an inflammatoryreflex stimulation module configured to repeatedly stimulate a patient'sinflammatory reflex and thereby inhibit the inflammation; a T-cellmodification module configured to administer a T-cell modifying agent toT-cells in a patient's spleen; wherein the T-cell modification moduleand the inflammatory reflex stimulation module communicate to coordinatestimulation of the inflammatory reflex and administration of the T-cellmodifying agent.
 15. The system of claim 14, wherein the T-cellmodification module comprises an implantable drug delivery device. 16.The system of claim 14, wherein the inflammatory reflex stimulationmodule comprises one or more electrodes configured to stimulate thevagus nerve and a controller configured to apply energy to one or moreelectrodes within a range sufficient to inhibit inflammation.